Vascular morphogenesis requires proper endothelial cell (EC) adhesion and migration. Transmembrane proteins tetraspanins are abundantly and ubiquitously present in endothelia. Our recent finding indicates that tetraspanin CD82 inhibits neovascularization in response to pathological stimuli. Our study also revealed that this tetraspanin inhibits neovascularization by restraining EC movement, restrains EC movement by confining EC adhesions, and confines EC adhesion by facilitating the endocytosis of cell adhesion molecules (CAMs) and preventing the aggregation of lipid rafts at the plasma membrane. The explicit and complete mechanisms that govern CD82-mediated inhibitions of pathological angiogenesis and EC movement, however, still remain largely unknown at the molecular, cellular, and organism levels. To elucidate how CD82 inhibits neovascularization, we hypothesize that, at the cellular level, CD82 down-regulates the dynamic EC-matrix adhesion, which is needed for proper EC movement. At the molecular level, CD82 reduces the functional cell adhesion proteins at the EC surface by altering the molecular landscape of membrane lipids and subsequently the endocytic machinery of ECs. In this project, we will first determine the mechanism by which CD82 selectively restrains pathological neovascularization. We will identify the CD82 effecter(s) that specifically affects pathological neovascularization, determine if CD82 confines angiogenic signaling that preferentially affects pathological neovascularization, and assess the effect of CD82 on the EC event(s) that mainly affects pathological neovascularization. Secondly, we will determine the mechanism by which CD82 alters the trafficking of cell adhesion molecules by examining the effects of CD82 on their endocytosis, recycling, and exosomal release in ECs. Finally, we will determine how CD82 organizes the membrane microdomains of ECs by assessing the activities of glycosphingolipid-metabolic enzymes upon CD82 removal, the regulatory effects of gangliosides on membrane microdomains, and the roles of gangliosides in CAM trafficking and in CD82- mediated inhibitions of EC movement and pathological angiogenesis. Thus, the goal of this project is to understand how CD82 selectively restrains pathological angiogenesis at the molecular, cellular, and organism levels. From these studies, we will delineate the mechanisms by which tetraspanins regulate vascular morphogenesis, establish a novel paradigm between pathological angiogenesis and membrane microdomain organization, and reveal the signaling axis that governs the crosstalk between EC movement and EC adhesion. From the in-depth mechanistic study, we will develop an integrated understanding of the unique features of CD82, which will ultimately lead to the development of therapeutic mean against pathological angiogenesis.